In vitro method for early or differential diagnosis or prognosis of myocardial infarction in a patient

ABSTRACT

The invention relates to a method for risk stratification for acute coronary syndrome (ACS), in particular acute myocardial infarction (AMI) and angina pectoris (AP), wherein provasopressin (proAVP) or fragments and partial peptides thereof, in particular copeptin or neurophysin II, is determined by an in vitro diagnosis.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/951,084, filed Jul. 25, 2013, which is a continuation of U.S.application Ser. No. 12/447,105, filed Sep. 22, 2009, now U.S. Pat. No.8,501,485, which is a national stage application (under 35 U.S.C. § 371)of PCT/DE2007/001928, filed Oct. 26, 2007, which claims benefit ofGerman application 102006050497.6, filed Oct. 26, 2006 and Germanapplication 102006057409.5, filed Dec. 4, 2006. The entire contents ofeach of these applications are hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

The invention relates to a method for risk stratification for acutecoronary syndrome (ACS), in particular acute myocardial infarction (AMI)and angina pectoris (AP), wherein provasopressin (proAVP) or fragmentsand partial peptides thereof, in particular copeptin or neurophysin II,is determined by an in vitro diagnosis. The invention further relates tosuitable combinations of biomarkers for in vitro diagnostics.

Risk stratification is becoming increasingly important for cardiacdisease in symptomatic as well as asymptomatic patients. In particularfor acute coronary syndrome there is a great need for suitable riskstratification.

Risk stratification is used to identify patients with an unfavorableprognosis, for intensive diagnostics, and for therapy/treatment, withthe objective of enabling an optimal clinical outcome. Accordingly, theaim of suitable risk stratification is to follow effective treatmentmethods for acute coronary syndrome, using percutaneous coronaryintervention and the newest medicaments.

Appropriate treatment requires early diagnosis and differentiation ofacute coronary syndrome, even upon emergency room admission, inconjunction with clinical decisions. Due to nonspecific symptoms such aschest pain in acute coronary syndrome, the differentiation anddelineation from other diseases as well as the identification of acutecoronary syndrome are essential.

In the prior art, the use of biochemical markers, in particularclassical markers such as cardiac troponin, myoglobin, and CK-MB mass,has been attempted for prognosis of myocardial infarction (Katus, H. A.;Remppis, A.; Scheffold, T.; Diederich, K. W., and Kuebler, W. (1991):Intracellular compartmentation of cardiac troponin T and its releasekinetics in patients with reperfused and nonreperfused myocardialinfarction, Am J Cardiol 67 (16): 1360-1367). Type B natriuretic peptide(BNP) together with pro-BNP (NT-ProBNP) (EP 1363128 B1, EP 1666881 A2)has proven to be another effective biochemical marker in myocardialdiagnostics.

Copeptin (also referred to as C-terminal proAVP) has been described inWO 2006/018315 (BRAHMS AG) as a biomarker for in vitro diagnosis of AMI.A corresponding copeptin assay is disclosed in Morgenthaler et al. (NilsG. Morgenthaler, Joachim Struck, Christine Alonso, and Andreas Bergmann,Assay for the measurement of copeptin, a stable peptide derived from theprecursor of vasopressin, Clinical Chemistry 52: 112-119, 2006).

Neurophysin has been described as a marker for nicotine uptake (RobinsonA. G., Isolation, assay, and secretion of individual human neurophysins,J Clin Invest 1975; 55: 360-367), malignancy- and nonmalignancy-relatedsyndrome of inappropriate ADH secretion (SIADH), and nephrogenicdiabetes insipidus (Pullan P. T., Clappison B. H., Johnston C. I.,Plasma vasopressin and human neurophysins in physiological andpathological states associated with changes in vasopressin secretion, JClin Endocrinol Metab 1979; 49: 580-587; North W. G., LaRochelle F. T.,Jr., Melton J., Mills R. C., Isolation and partial characterization oftwo human neurophysins: their use in the development of specificradioimmunoassays. J Clin Endocrinol Metab 1980; 51: 884-891).

However, a disadvantage of the known diagnostic methods using themarkers known heretofore is that early and complete identification ofat-risk patients is not achieved, and therefore risk stratification isinadequate. An object of the invention, therefore, is to develop amethod for risk stratification for acute coronary syndrome which allowsimproved identification of at-risk patients.

A further disadvantage is that in the prior art, sufficient sensitivityand/or specificity of the markers is usually not achieved.

A further object, therefore, is to provide a method for riskstratification for acute coronary syndrome, wherein at least one markeror a combination of markers has sufficient sensitivity and specificityin an in vitro diagnosis.

SUMMARY OF THE INVENTION

The object of the present invention, therefore, is to provide a methodfor risk stratification for acute coronary syndrome. The object isachieved by use of a method for risk stratification for acute coronarysyndrome, wherein provasopressin (proAVP) or fragments and partialpeptides thereof, in particular copeptin or neurophysin II, isdetermined by an in vitro diagnosis (referred to below as “methodaccording to the invention”).

Surprisingly, provasopressin (proAVP) or fragments and partial peptidesthereof, in particular copeptin or neurophysin II, has high sensitivityand specificity for the diagnosis of acute coronary syndrome (see theexamples and figures).

The term “acute coronary syndrome” encompasses various phases ofcoronary heart disease which are immediately life-threatening. Thisconcerns in particular emergency medical care, specifically, acutemyocardial infarction and/or angina pectoris, as well as sudden cardiacdeath. In addition to acute myocardial infarction, which according toWHO criteria (WHO (1979): Nomenclature and criteria for diagnosis ofischemic heart disease, Report of the Joint International Society andFederation of Cardiology/World Health Organization task force onstandardization of clinical nomenclature, Circulation 59 (3): 607-609)is defined as an acute chest pain event lasting longer than 20 minutesin conjunction with ST segment elevation and/or an increase inmyocardial enzymes, the term “unstable angina pectoris” (AP) has becomeestablished, which according to the invention is interpreted as “acutecoronary syndrome” (Hamm C. W.: Leitlinien: Akutes coronary syndrome(ACS)—Teil 1: ACS ohne persistierende ST-Hebung [Guidelines: Acutecoronary syndrome (ACS)—Part 1: ACS without persistent ST elevation], ZKardiol (2004) 93: 72-90; see also: Pschyrembel, De Gruyter, Berlin2004).

According to the invention, the term “risk stratification” encompassesthe identification of patients, in particular emergency care and at-riskpatients, with an unfavorable prognosis, for intensive diagnostics andfor therapy/treatment of acute coronary syndrome, in particularmyocardial infarction, and angina pectoris with the objective ofenabling an optimal clinical outcome. Risk stratification according tothe invention thus allows effective treatment methods for acute coronarysyndrome, using percutaneous coronary intervention and the newestmedicaments.

The invention therefore further relates to the identification ofpatients with increased risk and/or unfavorable prognosis of acutecoronary syndrome, in particular myocardial infarction and anginapectoris, and symptomatic and/or asymptomatic patients, in particularemergency care patients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of copeptin values in patients following myocardialinfarction. Plasma samples were collected from 131 patients, as stated,at various times following myocardial infarction. Copeptin values inhealthy subjects (controls; 200 values) are also plotted. Copeptinvalues for the groups are illustrated as box-and-whisker plots.

FIG. 2 shows a graph of copeptin values in patients following cardiacinfarction. Plasma samples were collected, as stated, at various timesfollowing myocardial infarction. The patients were then classified intogroups indicating whether death or rehospitalization due to cardiacinsufficiency subsequently occurred (“event”; 16 patients) or did notoccur (“surv”; 115 patients). Also illustrated are copeptin values inhealthy subjects (controls; 200 values). Copeptin values for the groupsare illustrated as box-and-whisker plots.

FIG. 3 shows the amino acid sequence of preprovasopressin (164 aminoacids) and the partial peptides and fragments of proAVP (amino acids: 29164), neurophysin II (amino acids: 32-124), and copeptin (amino acids:126-164).

DETAILED DESCRIPTION OF THE INVENTION

The method according to the invention may be used in a particularlyadvantageous manner to provide reliable stratification in emergencyand/or intensive medical care. The method according to the inventionthus allows clinical decisions resulting in rapid therapeutic successand avoidance of mortalities. Such clinical decisions also includefurther treatment with medicaments for treatment or therapy of acutecoronary syndrome, in particular acute myocardial infarction (AMI) andangina pectoris (AP).

The invention therefore further relates to a method for riskstratification in patients with acute coronary syndrome for makingclinical decisions such as further treatment and therapy withmedicaments, preferably in time-critical intensive or emergency medicalcare.

In one further preferred embodiment, the method according to theinvention therefore relates to therapeutic control of acute coronarysyndrome, in particular acute myocardial infarction (AMI) and anginapectoris (AP).

In one further preferred embodiment of the method according to theinvention, risk stratification is carried out for prognosis,differential diagnostic early detection and identification, severityassessment, and prognostic assessment in conjunction with therapy.

In one further preferred embodiment, the invention relates to a methodfor in vitro diagnostics for early or differential diagnosis orprognosis of myocardial infarction or angina pectoris, wherein themarker provasopressin (proAVP) or fragments and partial peptidesthereof, in particular copeptin or neurophysin II, is determined in apatient to be studied. However, copeptin or a fragment or partialsequence thereof, or neurophysin II or a fragment or partial sequencethereof, is particularly preferred.

The invention further relates to a method for risk stratification foracute coronary syndrome or a method for in vitro diagnostics for earlyor differential diagnosis or prognosis of myocardial infarction orangina pectoris according to one of the above embodiments, wherein afterthe appearance of symptoms a cutoff (threshold value) range of 6-20pmol/L of the marker provasopressin (proAVP) or fragments and partialpeptides thereof, in particular copeptin or neurophysin II, issignificant (specific) for the diagnosis and/or risk stratification.Also preferred is a cut-off (threshold value) of 6-20 pmol/L, inparticular 7.5 pmol/L, preferably up to 2 hours after the appearance ofsymptoms.

The invention further relates to a method for risk stratification foracute coronary syndrome or a method for in vitro diagnostics for earlyor differential diagnosis or prognosis of myocardial infarction orangina pectoris according to one of the above embodiments, wherein afterthe appearance of symptoms a cutoff (threshold value) range of 10-30pmol/L of the marker provasopressin (proAVP) or fragments and partialpeptides thereof, in particular copeptin or neurophysin II, issignificant (specific) for the prognosis and/or risk stratification.Also preferred is a cut-off (threshold value) of 10-20 pmol/L. On thisbasis these methods according to the invention are advantageouslysensitive.

In one embodiment of the method according to the invention, bodilyfluid, in particular blood, optionally whole blood or serum orcollectable plasma, is withdrawn from the patient to be studied, and thediagnosis is made in vitro/ex vivo, i.e., outside the human or animalbody. As a result of the determination of the marker provasopressin(proAVP) or fragments and partial peptides thereof, in particularcopeptin or neurophysin II, high sensitivity and specificity for acutecoronary syndrome, myocardial infarction, and angina pectoris areachieved, and diagnosis or risk stratification may be performed based onthe quantity present in at least one patient sample. However, the markercopeptin (stable fragment of proAVP or preprovasopressin) or a fragmentor partial sequence thereof is very particularly preferred. Alsoparticularly preferred is the marker neurophysin (stable fragment ofproAVP or preprovasopressin) or a fragment or partial sequence thereof.

Within the scope of the invention, “provasopressin” is understood tomean a human protein or polypeptide which may be obtained frompreprovasopressin, and for preprovasopressin includes amino acids 29-164(also see WO 2006/018315 and FIG. 3) and fragments or partial peptidesobtainable therefrom, in particular copeptin (fragment: amino acids126-164 (39 amino acids: SEQ: ASDRSNATQL DGPAGALLLR LVQLAGAPEPFEPAQPDAY) or neurophysin II (fragment: amino acids 32-124 ofpreprovasopressin (93 amino acids: SEQ: AMSDLELRQC LPCGPGGKGR CFGPSICCADELGCFVGTAE ALRCQEENYL PSPCQSGQKA CGSGGRCAAF GVCCNDESCV TEPECREGFH RRA).These polypeptides according to the invention may also havepost-translational modifications such as glycolization, lip(o)idization,or derivatization.

In one further preferred embodiment the invention relates to thediagnosis and/or risk stratification and/or early or differentialdiagnosis and/or prognosis for acute coronary syndrome, in particularacute myocardial infarction (AMI) and angina pectoris (AP), whereinneurophysin is determined in a patient to be studied.

In one further embodiment, provasopressin (proAVP) or fragments andpartial peptides thereof, in particular copeptin or neurophysin II, mayalso be determined using additional markers, preferably those whichalready indicate acute coronary syndrome, in particular myocardialinfarction or angina pectoris.

The invention therefore relates to such an embodiment of the methodaccording to the invention, wherein the determination is also carriedout using at least one additional marker selected from the group ofinflammatory markers, cardiovascular markers, neurohormonal markers, orischemic markers in a patient to be studied.

According to the invention, the inflammatory marker may be selected fromthe group comprising C-reactive protein (CRP), cytokines such asTNF-alpha, interleukins such as IL-6, procalcitonin (1-116, 3-116), andadhesion molecules such as VCAM or ICAM; and the cardiovascular marker,in particular a marker which indicates necrosis of cardiac muscletissue, and a marker which influences blood pressure, may be selectedfrom the group comprising creatin kinase, myoglobin, myeloperoxidase,natriuretic protein, in particular ANP (or ANF), proANP, NT-proANP, BNP,proBNP, or NT-proBNP, or in each case a partial sequence thereof,cardiac troponin, or CRP. Also included are circulation-regulating(pro)hormones, in particular progastrin-releasing peptide (proGRP),proendothelin-1, proleptin, proneuropeptide-Y, prosomatostatin,proneuropeptide-YY, proopiomelanocortin, proadrenomedullin (proADM), orin each case a partial sequence thereof. The ischemic marker may beselected from at least one marker from the group comprising troponin Iand T, and CK-MB. In addition, the neurohormonal marker may be at leastone natriuretic protein, in particular ANP (or ANF), proANP, NT-proANP,BNP, proBNP, or NT-proBNP, or in each case a partial sequence thereof.

In one particularly preferred embodiment, the invention relates to aparticularly advantageous combination of biomarkers and provasopressin(proAVP) or fragments and partial peptides thereof, in particularcopeptin or neurophysin II, with natriuretic proteins, ANP (or ANF),proANP, NT-proANP, BNP, proBNP, or NT-proBNP, or in each case a partialsequence thereof.

The invention therefore relates to a method for in vitro diagnosis ofacute coronary syndrome, myocardial infarction, or angina pectoris,wherein the marker provasopressin (proAVP) or fragments and partialpeptides thereof, in particular copeptin or neurophysin II, incombination with natriuretic proteins, in particular ANP (or ANF),proANP, NT-proANP, BNP, proBNP, or NT-proBNP, or in each case a partialsequence thereof, is determined in a patient to be studied. Once again,a combination of neurophysin II, copeptin, and BNP, proBNP, orNT-proBNP, in particular copeptin and proBNP, is particularly preferred.

It is particularly advantageous that the referenced biomarkercombinations have synergies which result in improved specificity andsensitivity for the diagnosis of the [ ]¹ (see examples). ¹Translator'snote: Apparent omission in source document.

In a further embodiment of the invention, for an in vitro diagnosis themethod according to the invention may be carried out by means ofparallel or simultaneous determinations of the markers (for example,using multititer plates containing 96 or more cavities), wherein thedeterminations are carried out for at least one patient sample.

In addition, the method according to the invention and determinationstherefor may be carried out in a diagnostic device based on an automaticanalyzer, in particular using a Kryptor system(http://www.kryptor.net/).

In a further embodiment, the method according to the invention anddeterminations therefor may be carried out using a rapid test (forexample, a lateral flow test) in either single- or multi-parameterdeterminations. One particularly preferred embodiment involves aself-test, or a device which is suitable for emergency care diagnostics.

The invention further relates to the use of provasopressin (proAVP) orfragments and partial peptides thereof, in particular copeptin orneurophysin II, for risk stratification for acute coronary syndrome,myocardial infarction, or angina pectoris, and/or for in vitrodiagnostics for early or differential diagnosis or prognosis ofmyocardial infarction or angina pectoris.

In one special embodiment the invention relates to the use ofprovasopressin (proAVP) or fragments and partial peptides thereof, inparticular copeptin or neurophysin II, in combination with natriureticproteins, in particular ANP (or ANF), proANP, NT-proANP, BNP, proBNP, orNT-proBNP, or in each case a partial sequence thereof, for diagnosisand/or risk stratification for acute coronary syndrome, myocardialinfarction, or angina pectoris.

A further object is to provide a corresponding diagnostic device forcarrying out the methods according to the invention.

Within the scope of the invention, such a diagnostic device, inparticular an array or assay (for example, immunoassay, ELISA, etc.), isunderstood in the broadest sense as a device for carrying out themethods according to the invention.

The invention further relates to a kit for risk stratification for acutecoronary syndrome, myocardial infarction, and/or angina pectoris,containing detection reagents for determining provasopressin (proAVP) orfragments and partial peptides thereof, in particular copeptin orneurophysin II, and optionally the additional markers stated above. Suchdetection reagents include antibodies, for example.

In one special embodiment the invention relates to a kit for diagnosisand/or risk stratification for acute coronary syndrome, myocardialinfarction, and/or angina pectoris, containing detection reagents fordetermining provasopressin (proAVP) or fragments and partial peptidesthereof, in particular copeptin or neurophysin II in combination withnatriuretic proteins, in particular ANP (or ANF), proANP, NT-proANP,BNP, proBNP, or NT-proBNP, or in each case a partial sequence thereof,and optionally the additional markers stated above. Such detectionreagents include antibodies, for example.

The following examples and figures are used for a more detailedexplanation of the invention, but do not limit the invention to saidexamples and figures.

EXAMPLES AND FIGURES

Patients admitted into emergency care of a hospital with the primarysymptom of chest pain had a blood sample taken during the initialexamination.

131 patients with myocardial infarction (MI) (median age: 64.0 years)were observed for the appearance of acute MI symptoms over a totalperiod of 180 days:

Within the first 6 hours after appearance of symptoms (chest pain) andadmittance to the hospital, 3 blood withdrawals were made, one each at0-2 hours, 2-4 hours, and 4-6 hours after appearance of MI symptoms.

Copeptin and NT-ProBNP were determined in each case.

After hospitalization, within the first 4 days after therapeuticintervention (standard, see Braunwald et al., 2002) (day 2-5), in eachcase one blood withdrawal with subsequent copeptin determination wasperformed.

After dismissal (median hospital stay: 7.2 days), the patients wereobserved over a period of 180 days. The development of severe cardiacsufficiency and/or death of the patient were determined to be events. Ofthe observed patients, 115 had no events (o.E.) and 16 had events (+E.).The median ages of the groups were 63.8 and 64.5 years.

For evaluating the diagnostic effectiveness of the biomarkers for theearly diagnosis of MI and for risk stratification, the results fromindividual patient blood withdrawals were compared to those from 200individuals (median age: 65.2 years) who showed no symptoms of MI(controls). The results are shown in Table 1 and FIGS. 1 and 2.

Table 1 shows the sensitivities (values>cutoff (threshold value)) for MIpatients compared to the corresponding specificities (values<cutoff(threshold value)) for the controls at the indicated times afterappearance of symptoms.

Within the earliest observation period (0-2 hours) the copeptin wasgreatly elevated compared to the controls. For example, only 4.5% of allcontrols exhibited a copeptin value greater than 7.5 pmol/L (examplecutoff (threshold value)) (specificity=95.5%), whereas 78.3% of thepatients without subsequent events, and even 87.5% of the patients withevents, exhibited copeptin concentrations greater than 7.5 pmol/L(sensitivity). Compared to the markers used thus far in routinediagnostics (troponin, myoglobin, CK-MB), this resulted in surprisingincreases in the sensitivities at points in time which then allowedthese biomarkers to be used for early or differential diagnosis of MI,as well as for risk stratification for acute coronary syndrome(troponin; CK-MB generally appeared only after 6 hours post-event, whilemyoglobin appeared early with less sensitivity, but then dropped againapproximately 2 hours later; i.e., resulted in an uncertain diagnosticwindow). Advantageously, the increase in copeptin, i.e., the time of theblood withdrawal, was of secondary importance for early diagnostics (atthe 7.5 pmol/L cutoff, for example, 78.3/80.0/73% sensitivity at0-2/2-4/4-6 hours, respectively).

Combination with proBNP or NT-ProBNP

Surprisingly, it was shown that the combination of copeptin with proBNP(NT-ProBNP) allowed a marked increase in the sensitivity of earlydiagnosis of MI (see Table 1). Although proBNP (NT-ProBNP) was inferiorto the diagnostic effectiveness of copeptin, for the evaluation ofcopeptin>7.5 pmol/L and/or proBNP (NT-ProBNP)>200 pmol/mL, for example,at a specificity of approximately 95% which was comparable to thecorresponding individual markers, it allowed an increase of 3-6% in thesensitivities at all times in the non-event group, and in the eventgroup, an increase of 6-9.5% compared to the sensitivities of copeptinas an individual marker.

TABLE 1 0-2 h 0-2 h 2-4 h 2-4 h 4-6 h 4-6 h Controls Copeptin (pmol/L)o.E. +E. o.E. +E. o.E. +E. (specificity) Sens/Spef 88.7% 93.8% 89.6%93.8% 80.0% 87.5% 93.0% for cutoff 6 Sens/Spef 78.3% 87.5% 80.0% 87.5%73.0% 75.0% 95.5% for cutoff 7.5 Sens/Spef 67.0% 68.8% 68.7% 68.8% 56.5%68.8% 97.5% for cutoff 10 Sens/Spef 44.3% 8.8% 43.5% 68.8% 40.0% 62.5%100.0% for cutoff 20 NT-ProBNP (pmol/mL), 58.5% 62.0% 61.5% 65.0% 66.5%70.0% 94.5% cutoff 200 Combination: 81.2% 93.5% 83.5% 96.0% 79.5% 84.5%93.8% copeptin >7.5 pmol/L and/or NT-ProBNP >200 pmol/mL

Sample recovery, biomarker analysis:

Blood withdrawals were made using standard serum monovettes. After aclotting time of 20-40 min, centrifugation was performed for 15 min at2000 g, followed by serum separation by decanting. The serum sampleswere stored at −20° C. for further use.

NT-ProBNP was determined according to Omland et al. (Omland T., PerssonA., Ng L., et al., N-terminal pro-B-type natriuretic peptide andlong-term mortality in acute coronary syndromes, Circulation, 2002; 106:2913-2918) using a luminescence immunoassay.

Copeptin was determined using the copeptin luminescence immunoassay fromBRAHMS AG. The copeptin determination method is described in detail inMorgenthaler N. G. et al., Clin. Chem. 2006 January, 52 (1), 112-119. Insummary: a 50-microliter sample was pipetted into a test tube coatedwith copeptin antibody (AB1) and mixed with 200 microliters of asolution of acridinium ester-marked anti-copeptin antibody (AB2) andincubated for 2 hours at room temperature. After removal of unbound(free) marked antibody by washing four times with wash solution(Lumitest wash solution, Brahms A G), bound acridinium ester-markedantibody was determined in a luminometer from Berthold.

Prognosis (Prediction of Event Occurrence):

Surprisingly, it was shown that MI patients with events within the 180days post hospitalization exhibited even higher copeptin concentrationsthan patients without events. Within 6 hours after appearance of MIsymptoms, at the example cutoff (threshold value) of 30 pmol/L copeptinshowed a relative risk of approximately 1:2 (patients with copeptin>30had twice the risk for subsequent events compared to patients<30pmol/L).

As expected, the concentrations decreased after the intervention (day2ff., Table 2b). In this case it was surprisingly shown that patientswith subsequent events exhibited a much lower reduction in thebiomarker. This led to a sharp increase in risk, to values of up to1:7.14 for patients with a copeptin value higher than 30 pmol/L.

TABLE 2a 0-2 h 0-2 h 2-4 h 2-4 h 4-6 h 4-6 h Copeptin +E. o.E. +E. o.E.+E. o.E. Patients >30 pmol/L 62.5% 36.5% 62.5% 35.7% 62.5% 27.8%Relative risk for cardiac 1.7 1.75 2.25 insufficiency or death up to 180days after dismissal from the hospital

TABLE 2B Day 2 Day 2 Day 3 Day 3 Day 4 Day 4 Day 5 Day 5 Copeptin +E.o.E. +E. o.E. +E. o.E. +E. o.E. Patients >30 pmol/L 50.0% 7.0% 43.8%7.0% 39.2% 6.1% 31.3% 6.1% Relative risk for cardiac 7.14 6.26 6.42 5.13insufficiency or death up to 180 days after dismissal from the hospital

Legend in tables and figures: surv.=survival, E.=event: death,development of severe cardiac insufficiency, cutoff=threshold value inpmol/L, Sens=sensitivity, Spef=specificity, o.=without.

Neurophysin:

A radioimmunoassay for neurophysin was prepared according to earlierdescriptions (Pullan (supra)): neurohypophysis neurophysin was isolatedand quantified. In this manner rabbits were immunized, resulting inrecovery of high-titer anti-neurophysin antisera. The highest-titerantiserum was used in a concentration of 1:100,000 for the immunoassay.Purified neurophysin was radioiodized using the chloramine T method andused as a tracer in the assay. Dilutions of purified neurophysin instandard equine serum were used as standards. The assay was carried outas follows: A 50-μL sample or standard was mixed with 100 μL tracer(12,000 dpm per determination) and 100 μL diluted anti-neurophysinantiserum, and incubated for 24 hours at 4° C. 100 mM sodium phosphate,pH 7.5, and 0.1% BSA were used as buffer. An antibody-bound tracer wasseparated from free tracer by adding 60% ethanol and then centrifugingfor 15 minutes at 4° C. and 5,000 g. The supernatant was discarded, andthe residual radioactivity in the pellet was determined. The analysiswas performed using Multicalc software. The assay had an analyticaldetection limit of 22 pg/mL. The assay had a measurement range up to 400pg/mL. The assay was used to measure plasma samples from variouspatients as described below. Samples with measured values>400 pg/mL weremeasured in suitable dilutions, resulting in measured values within themeasurement range.

Myocardial Infarction/Diagnosis

In 66 patients with acute cardiac infarction, samples were taken nolater than 6 hours after occurrence of the cardiac infarction, andneurophysin was measured. For purposes of comparison, neurophysin wasdetermined in 200 healthy controls. The receiver operatingcharacteristics analysis for diagnosis of cardiac infarction resulted inan AUC of 0.95. For a cutoff value of 213 pg/mL a sensitivity of 62.6%was obtained at a specificity of 98%. For a cutoff value of 136.1 pg/mLa sensitivity of 84% was obtained at a specificity of 95%.

Myocardial Infarction/Prognosis

In 66 patients with acute cardiac infarction, samples were taken nolater than 6 hours after onset of the cardiac infarction or on thesecond day after the infarction, and neurophysin was measured. Thepatients were observed for a period of 360 days. During this period 58patients had no adverse event, and 8 died or were rehospitalized due tocardiac insufficiency.

Prognosis for Day 1 (<6 Hours After Occurrence of the Infarction):

Receiver operating characteristics analysis was used to determine thebest cutoff value (defined as the largest product of the sensitivity andspecificity) for prognosis of mortality or rehospitalization due tocardiac insufficiency: 777 pg/mL. At this cutoff value the sensitivityof the prognosis was 62.5%, and the specificity was 73%. The likelihoodratio of an adverse event at a cutoff value of 777 pg/mL was 2.3.

Prognosis for Day 2:

Receiver operating characteristics analysis was used to determine thebest cutoff value (defined as the largest product of the sensitivity andspecificity) for prognosis of mortality or rehospitalization due tocardiac insufficiency: 261 pg/mL. At this cutoff value the sensitivityof the prognosis was 68.8%, and the specificity was 73%. The likelihoodratio of an adverse event at a cutoff value of 261 pg/mL was 2.6.

The invention claimed is:
 1. An in vitro method for differentialdiagnosis or prognosis of myocardial infarction in a patient, saidmethod comprising: determining in at least one sample of bodily fluidfrom a patient to be studied the level of copeptin and neurophysin II,wherein said at least one sample of bodily fluid is taken from saidpatient within the first 6 hours after appearance of symptoms of acutecoronary syndrome or myocardial infarction, wherein a threshold level ofcopeptin of 6-20 pmol/L and a threshold level of neurophysin II of 6-10pmol/L are indicative of differential diagnosis or prognosis ofmyocardial infarction.
 2. The method according to claim 1, furthercomprising determining the level of a natriuretic peptide, or a partialsequence thereof in said at least one sample of bodily fluid from saidpatient.
 3. The method according to claim 2, wherein said natriureticpeptide is ANP (or ANF), proANP, NT-proANP, BNP, proBNP, or NT-proBNP,or partial sequences thereof.
 4. The method according to claim 3,wherein said natriuretic peptide is proBNP.
 5. The method according toclaim 4, wherein a threshold level of 200 pmol/L of proBNP is indicativeof an increased risk of myocardial infarction.
 6. The method accordingto claim 1, wherein said threshold level is 7.5 pmol/L of neurophysinII.
 7. The method according to claim 1, further comprising determiningthe level of at least one marker selected from the group consisting ofinflammatory markers, cardiovascular markers, neurohormonal markers, andischemic markers in said at least one sample of bodily fluid from saidpatient.
 8. The method according to claim 7, wherein said at least onemarker is selected from the group consisting of C-reactive protein(CRP), cytokines, interleukins, procalcitonin (1-116, 3-116), andadhesion molecules.
 9. The method according to claim 7, wherein said atleast one marker is selected from the group consisting of creatinekinase, myoglobin, myeloperoxidase, a natriuretic peptide or partialsequence thereof, cardiac troponin, C-reactive protein, andcirculation-regulating (pro)hormones.
 10. The method according to claim7, wherein said at least one marker is selected from the groupconsisting of troponin I and T, and CK-MB.
 11. The method according toclaim 1, wherein said threshold level is 7.5 pmol/L of copeptin.
 12. Themethod according to claim 1, wherein said at least one sample of bodilyfluid is taken from said patient within 2 hours after the appearance ofsymptoms of acute coronary syndrome or myocardial infarction.